BRPI0607794B1 - mixing at least 6 species of lactic acid bacteria and / or bifidobacteria in sour dough manufacturing - Google Patents

Abstract

mixing at least 6 species of lactic acid bacteria and / or bifidobacteria in sour dough making. The present invention relates to a mixture of at least 6 species of lactic acid bacteria and / or bifidobacteria that is described for use in baking and the medical field. The preferred mixture comprises streptococcus thermophilus, infant bifidobacter, bifidobacterium longum, bifidobacterium brevis, lactobacilius acidophilus, lactobacilius plantarum, lactobacilius casei, lactobacilus detbrueckii subsp. Bulgaricus. Said mixture is useful for sour dough, fermentation composition. cooked articles and other food products obtained therefrom are described. These articles have a low or no gluten content and are suitable for the dietary integration of a patient suffering from sciatica, to reduce the risk of allergies due to wheat flour albumin and globulin for the treatment of schizophrenic symptoms, in the preparation of enteral rich diet products.

Description

Report of the Invention Patent for "MIXING OF AT LEAST 6 SPECIES OF LACTIC ACID BACTERIA AND / OR BIFIDOBACTERIA IN THE MANUFACTURE OF SUGAR PASTA". The present invention relates to the manufacture of cooked articles and generally to starchy food. Eia provides cooked foods and other foods - which are more digestible, they are gluten free or have a reduced and markedly hydrolyzed gluten content and are particularly suitable for celiac patients.

Background of the Invention Cereals are important components of the daily diet. However, wheat flour gluten, and in particular the gliadin fraction, are responsible for human intolerance. Celiac disease, also known as sprue celiac (CS) or gluten-sensitive enteropathy, is a widespread food intolerance that occurs in 1 in 130 to 300 people in European and North American populations. In South America, North Africa and Asia is generally underestimated (Fasano and Catassi. 2001, Gastroentero-fogy, 120: 636-651). The epidemiological distribution of SC is effectively conceptualized by the iceberg model introduced by Logan in 1992 (Logan; 1992, Dyn. Nutr. Res. 2: 14-24) where the prevalence of the disease is influenced by the frequency of predisposition genotypes in the population. Completely and forever avoiding gluten ingestion remains the cornerstone of CS treatment. The International Food Authority has now redefined the term gluten free to zero gluten tolerance, while Codex Ali-mentarius allows a concentration of 2000 ppm gluten per food. Efforts to reduce human intolerance to cereals are of great medical, nutritional and economic interest. This is particularly true in the current context where bakery industries are using very fast technological processes that can influence the expansion of CS epidemiology.

So the need for more digestible and tolerated bread and baked goods is really felt. CS is an autoimmune disease of the small intestine mucosa in genetically susceptible people. Upon ingestion of gluten, these patients suffer from self-perpetuating mucosal inflammation characterized by progressive loss of villi and crypt hyperplasia (Silano and De Vicenzi, 1999; Nahrung, 43: 175-184). During enoluminal proteolytic digestion, for example, wheat gliadins release a family of Pro and Glu-rich oligopeptides that are responsible for T-cell mediated immunoresponse and / or, more generally, the inflammatory state that characterizes the state. CS (Silano and De Vicenzi; 1999). The literature reports the identification of the following oligopeptides: A-gliadin fragments 31-43 (Silano and De Vicenzi; 1999), a2-gliadin fragment 62-75 (Auricchio, S., et al.; 1996, Scand. J. Gastroenterol., 31: 247-253; Picarelli, A., et al., 1999, Scand. J. Gastroenterol., 34: 1099-1102), epitope 33-mer, which corresponds to fragment 57-89 of α2-gliadin (Shan, L., et al.; 2002. Science, 297: 2275-2279), γ-gliadin fragment 134-153 (Aleanzi, M., et al., 2001, Clin. Chem., 47: 2023- 2028) and α9-gliadin fragment 57-68 (Arentz-Hansen, et al.; 2000, J, Exp. Med., 191: 603-612). Flours that are not tolerated by CS patients included wheat, rye, barley, kamut, triticale and spelled. There is some controversy still debated over oats.

Multidisciplinary research efforts are carried out in various fields to administer CS. They are concerned with engineering gluten-free grains (Fasano, A. et al.; 2003, Arch. Intern. Med., 163: 286-292), researching CS genes in humans (Fasano, A., et al.; 2003), the use of some protective substances such as mannans and N-acetylglycosamine oligomers and the use of a bacterial Fiavobacterium meningosepticum prolyl endopeptidase as an oral supplemental therapy (Shan, et al .; 2002).

More recently, two articles have shown extensive hydrolysis of gliadin fractions by sour-selected lactobacilli, such as LactobaciUus alimentaryius 15M, L. Brevis 14G, L. sanfranciscensis 7A and L. hitgardii 51B (Di Cagno, et al.; 2002 (Appl. Environ. Microbiol., 68: 623-33) and especially the tolerance of 17 patients with SC to breads containing 2 g of gluten as determined by in vivo challenges based on intestinal permeability (Di Cagno, et al. 2004, Appl Environ Microbiol., 70: 1088-1096). These results, while encouraging, at least in view of their symptomatology, did not show regression of histopathological damage.

Wei et al. (Weí, J. and Hemmings, GP; Medical Hypotheses, (2005), 64, 547-552) establish a genetic relationship between celiac disease and schizophrenia, and report the beneficial effect of regression of schizophrenic symptoms in treated celiac patients. on a gluten-free diet (De Santis, A., et al.; J. Intern. Med. 1997; 242: 421-3). The use of certain lactic acid bacteria in the manufacture of cooked foods is also known. US 4,140,800, from Kline, describes a process for preparing a lyophilized sour dough baking starter composition using Lactobacillus sanfrancisco to provide a useful product in the preparation of French bread. Desirably, the flour is high in gluten. The solution provided by Di Cagno et al. Of using lactic acid bacteria for sour dough still leaves some unresolved practical problems. In particular, (i) selected strains are the time consuming research results that showed marked differences in strain level within species above lactic acid bacteria for sour mass; (ii) the same results are obviously not reproducible in the bakery factory; and especially (iii) the selected strains are not commercially available.

Other references describe the use of lactic acid bacteria and bifidobacteria in food manufacturing. EP 0 856 259 relates to a food composition containing a mixture of lyophilized live bacteria comprising at least two species of bacteria selected from bifidobacteria and at least two species of bacteria selected from Lactobacillus acidophi-lus, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum and Streptococcus faecium and one or more oligosaccharides. The composition is added to a liquid, creamy or pasty food, wherein said food product is milk, milk-based or milk-derived product, or a vegetable-based or derived product, the supplementation being performed at the time. of the use of the food product. The product is not used in baking. WO 03/071883 relates to dietary and / or pharmaceutical compositions for humans and / or animals, and general food products based on microbial cultures consisting of autochthonous and allochthonous species with respect to humans and animals. , selected from lactic acid bacteria, propionibacteria, yeast and / or other molds. They have a balancing action on the intestinal flora of the human host or animal, as well as have several beneficial / probiotic effects with respect to the host organism. There is no indication of a possible use in celiac disease. US 2004/265291 provides compositions, kits, and methods for providing or recovering beneficial bacteria for a patient. The compositions and kits optionally include food or nutrients that promote the growth and proliferation of bacteria in the patient or an antimicrobial agent to reduce the presence of undesirable or pathogenic microbes in the patient. WO 02/065842 relates to starter preparations suitable for all types of cereal and their use to produce bread and baking products based on yeast or using yeast, especially to produce gluten-free bakery products for people with celiac disease. There is no description of particular mixtures of lactic acid bacteria and bifidobacteria. US 5,185,165 discloses a precursor base for use in dough product comprising an acid concentrate, at least one type of sugar, yeast, at least one type of flour, non-fat dry milk and at least one type of bacterium. lactic acid producer and a process for producing the precursor base. The precursor base is useful in a process for producing a precursor paste (or active yeast concentrate) for use in making a pre-fermented dough mix of the bread dough product. In addition, processes for preparing the dough are described. precursor mixture and the pre-fermented dough mix and an apparatus for producing the pre-fermented dough mix. The reference to lactic acid bacteria is totally generic. US 2004/110270 describes a bacterial composition having immunomodulatory properties comprising at least one strain selected from the group consisting of Lactobacillus acidophilus PTA-4797, LactobaciHus piantarum PTA-4799, Lactobacillus salivarius PTA-4800, Lac-tobacillus paracasei PTA-4798 bifidum PTA-4801 and Bifido-bacterium lactis PTA-4802. EP 1 258 526 describes the production of an initiator for making wheat premeal and sour wheat pasta by partially fermenting a mixture of water and ground wheat product (s) with an inoculum comprising lactobacilli and yeast comprising the use of an inoculum containing an adapted mixed flora comprising at least one yeast strain, at least one homofermentative lactobacillus strain and at least one heterofermentative lactobacillus strain. Strains of Saccharomyces sp. DSM 14265, Lactobacillus pontis D SM 14269, Lactobacillus pontis DSM 14272, Lactobacillus pontis DSM 14273, Lactobacillus pontis 14274, LactobaciHus crispatus DSM 14271, LactobaciHus piantarum DSM 14268 and Lactobacillis sanf; an adapted mixed flora comprising Saccharomyces sp. DSM 14265 and at least three of Lactobacillus pontis DSM 14269, Lactobacillus pontis DSM 14272, Lactobacillus pontis DSM 14273, Lactobacillus pontis DSM 14274, Lactobacillus crispatus DSM 14271 and Lactobacillus piantarum. bakery products without special drug indications. WO 99/09839 refers to a paste-like composition that is applicable for use as such and as a filler, topping or other component of various food products, and which contains a significant amount of probiotic. The food product is preferably a bakery product, in particular a rye bread, cookie, breadcrumbs or the like. This reference deals with the generally known aspects of using probiotics. The need for a readily available cooked product for celiac disease patients, a reproducible manufacturing process and reliable, safe and commercially available materials is still felt in this field. The present invention fulfills these needs by providing a baked product for patients affected by celiac disease. However, the cooked product has great utility in the human diet due to its higher digestibility.

Summary of the Invention It has been found that certain specific mixtures of lactic acid bacteria and bifidobacteria of human origin or milk have the surprising property of being able to hydrolyze fractions of gliadin and gfutenin which are responsible for celiac disease.

These specific mixtures are quite useful in making sour dough and provide well-defined bacterial species.

Therefore, it is an object of the present invention to use bacterial specific mixtures of lactic acid and bifidobacteria in the manufacture of sour dough.

An object of the present invention is represented by cereal based food, in particular cooked articles which are generally more digestible and in particular can be tolerated by patients with CS.

Another object of the present invention is a method for making cereal-based food, in particular baked articles suitable for patients affected with celiac disease and suitable for preventing gluten contamination in gluten-free products.

Another object of the present invention is gluten-free cereal-based food, in particular baked products made from wheat flour when the use of specific mixtures of lactic acid bacteria and bifidobacteria is implemented under specific conditions with microbial proteolytic enzymes. routinely used in the baking industry.

Another object of the present invention is a food for patients affected by celiac disease, wherein said food contains the specific mixture of lactic acid bacteria and bifidobacteria described herein.

Still another object of the present invention is the use of the aforementioned mixtures of lactic acid bacteria and bifidobacteria for the preparation of a useful Plaque-Activating Factor (PAF) reducing product and other inflammatory cytokines.

These and other objects of the present invention will now be described in detail also by way of examples and Figures, wherein: Figure 1 shows the 2DE analysis of gliadin protein fractions of different pasta made from wheat flour. (A) Chemically acidified mass (control). Prolamine polypeptides were indicated by numbered red ovals. (B) Mass incubated for 24 hours at 37 ° C with MIX 1 of Example below. Prolamine polypeptides were indicated by numbered red ovals. Blue numbers refer to polypeptides that have been degraded by more than 80%. Mr, molecular weight.

Figure 2 shows hydrolysis of 33-mer peptide by MIX 1 (109 CFU / mL). 214 nm UV RP-FPLC, 200 μ 200 traces of 33-mer after 24 h incubation at 37 ° C without microbial inoculum (A) and after 24 hours of hydrolysis by MIX 1 at 37 ° C (B) .

Detailed Description of the Invention According to the present invention, a mixture of at least 6, preferably at least 7, more preferably at least 8 species of lactic acid bacteria and / or bifidobacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacillus ceiiobiosus, Lactobacillus críspatus, Lactobacillus curvatus, Lactobacillus deibrueckii, Lactobacillus deibrueckii subsp. bulgaricus, Lactobacillus deibrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus Leich-mannii, Lactobacillus minutus, Lactobacillus paracasei, Lactobacillus plant rum, Lactobacillus rogosae, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus gasseri, Lactobacillus fermentum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve , Bifidobacterium catenuiatum, Bifidobacterium dentium, Bifidobacterium erik-sonii, Children's Bifidobacterium, Bifidobacterium iactis, Bifidobacterium iongum, Bifidobacterium pseudo-catenulatum, Bifidobacterium pseococcus, Ferococcus pseudococcus, Ferococcus pseudococcus -lulomonas fermentans, Zymomonas mobilis, Streptococcus thermophilus are suitable for the present invention.

Other species may be used, for example, those described in the prior art and generally available from collections such as ECACC, ASTIM; DSM

Preferred mixtures according to the present invention are as follows: Streptococcus thermophilus, Infant Bifidobacterium, Bifidobacterium Iongum, Bifidobacterium brevis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, subsp. Bulgari-cus.

Streptococcus thermophilus, Bifidobacterium Iactis, Bifidobacterium brevis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus case, Lactobacillus helveticus.

These mixtures of well-known species can easily be prepared by anyone having ordinary knowledge in this field.

Conveniently, such mixtures are commercially available in lyophilized form.

These formulations are suitable for use as a starter in the preparation of sour dough. Cereal-based food, in particular cooked foods obtained according to the present invention are generally more digestible, therefore they are more accepted by the general consumer or particularly by people who desire or need more digestible food.

In a particular embodiment of the invention, cereal-based food, in particular cooked foods may be used for the integration of the diet of people affected by celiac disease, as the gluten concentration is reduced to a low value and the amount of gluten that has persisted in the mass is markedly hydrolyzed, especially for the polypeptide sequences that are responsible for CS.

When one of the above microbial mixtures is integrated with a sufficient amount of microbial protease, such as, for example, 200 ppm microbial protease (typically from Aspergillus sp.) Under the conditions optimized in the present invention, the fermented sour dough has a concentration of less than 200 ppm as determined by the use of monoclonal antibody R5. As established by Codex Alimentari-us, this type of product is defined as gluten free and therefore suitable for celiac patients.

Microbial proteases are commonly used in baking, see, for example, WO 88/03365, EP 0588426, US 6,465,209, GB 1,196,946. Such proteases are commonly marketed, see, for example, Enzyme Deveriopment Corporation (US), and the present invention may be carried out with any commercially available bakery product.

In a preferred embodiment of the present invention, the microbial protease is a fungal Aspergillus oryzae protease activity of 500,000 HUT / g; optimal pH of about 3.0 and activity in the pH range 3.0 to 6.0; the optimum temperature of about 50 ° C and activity in the range of 25 to 60 ° C; or another protease is an Aspergillus niger acid stable protease, activity of 3,000 SAPU / g; optimal pH 2.0-3.0 and activity in the pH 2.0 to 6.0 range; optimum temperature of 50-60 ° C and activity in the range of 30-60 ° C. These enzymes are available from Bio-Cat Inc., Troy, Virginia, USA and many other suppliers. The present invention allows the manufacture of baked articles with a higher percentage of wheat flour, resulting in a product with a more pleasant aroma and better acceptance by people affected by celiac disease. The present invention also makes it possible to obtain products directed to the general consumer, including healthy people, with more digestibility.

In a broader aspect, the present invention also relates to starch products which comprise a mixture of lactic acid bacteria, optionally provided with enzyme preparations as described above.

In this broader aspect, the present invention provides a mixture of lactic acid bacteria and bifidobacteria, optionally added proteolytic enzymes of microbial origin, useful for the preparation of products for oral administration to improve digestion of gluten and gluten related substances. The sour dough comprising the specific mixture of the present invention is the critical aspect thereof. Sour dough is useful in a process for the preparation of a baked article, in particular bread, but this applies to all fermented and unfermented products such as cookies, pastries, cakes, pies, pizza, crackers. , breadsticks, snacks and all other products known in the art. The sour dough according to the present invention is additionally suitable for preparations for making also home-made cereal-based foods, in particular baked goods. In that case, the package for a baked article will comprise, in addition to the ingredients used for the specific product, a fermentation preparation comprising the specific mixture of the present invention. The fermentation preparation according to the present invention may be a combination with the specific mixture of lactic acid bacteria and bifidobacteria or may be provided in the package separately with the mixture of lactic acid bacteria and bifidobacteria and may be mixed with the latter. , at the time of use, for example, in water to form a fermentation suspension. The lactic acid bacterial mixture may be packaged in a container alone or in admixture with the proteolytic enzymes (protease) discussed above.

Starchy products are generally well known in the field and are common knowledge, also among consumers and in home cooking. In particular, the present invention is applied to cereal products.

Examples of starch products are all types of pasta, noodles such as noodles, instant fry noodles and wet noodles, snack products, tortillas, corn slices, extruded cereals and stripped cereals. Methods for making pasta are well known in the art and should be referred to simply, for example, Pasta and Semolina Technology, Edited by R.C. Kill and K. Turnbull, Blackwell Science, 2001 and Barilla patents. Methods for making Asian starch products are also well known and a simple exemplary reference is made to Asian Food, Science and Technology, edited by Catharina YW, Ang, KeShun Liu and Yao-Wen Huang, Technomic Publishing Company, Inc., 1999. and US 20020160093 by Kao Corporation and WO 99/65331 of So-cieté de Produites Nestlé SA

Most pastes today are made by high-capacity continuous extruders operating on the drill extrusion principle where kneading and dough extrusion includes production of dry noodles, noodles and spaghetti.

Pasta products are made by mixing ground wheat, water, eggs (eg egg noodles or egg spaghetti), and sometimes optional ingredients. These optional ingredients are typically added to a high capacity continuous drill extruder which can be equipped with a variety of dough forming dies. The dough is then dried and packaged for the market.

Pasta products contain ground wheat, water, and occasionally eggs and / or optional ingredients. Pasta makers typically use ground durum wheat (semolina, durum granules and durum flour) in pasta production, although fine flour and common wheat flour are occasionally used. Most pasta manufacturers prefer semolina, which consists of fine particles of uniform size and produces the highest quality pasta product. Water used in pasta production should be pure, free of unpleasant odors, and suitable for drinking. Also, since pasta is produced below pasteurization temperatures, water should be used with low bacterial counts. Eggs (fresh eggs, frozen eggs, dried eggs, egg yolks, or dried egg solids) are added to the dough to make egg noodles or egg spaghetti and to improve the nutritional quality and richness of the paste. Small amounts of optional ingredients, such as salt, celery, garlic and bay leaves, can also be added to the dough to improve flavor. Disodium phosphate can also be used to reduce cooking time. Other ingredients such as gluten gum, glyceryl monostearate and egg whites may also be added. All optional ingredients should be clearly labeled on the packaging. Durum wheat is ground into semolina, durum granule, or durum flour using roller mills. The grinding of semolina is unique because the goal is to prepare medium granuars with a minimum of flour production. Once ground, the wheat is mixed with water, eggs, and any other optional ingredients.

In the mixing operation, water is added to the milled wheat in a mixing chute to produce the pasta with a moisture content of approximately 31%. Eggs and any optional ingredients may also be added. Most modern dough presses are equipped with a vacuum chamber to remove air bubbles from the dough before extrusion. If air is not removed prior to extrusion, small bubbles will form in the mass that decrease mechanical strength, yielding a chalk-white finished product.

Once the dough has been mixed, it is transferred to the extruder. The extruder drill not only forces the dough through the die, it also kneads the dough to form a homogeneous dough, controls the production rate, and influences the overall quality of the finished product. Although the construction and size of extrusion drills vary with equipment manufacturers, most modern presses have sharp edge drills that have a uniform pitch over their entire length. The drill fits into a slotted extrusion barrel, which assists the dough to move forward and reduces friction between the drill and the inside of the barrel. Extrusion barrels are equipped with cooling liners to dissipate heat generated during the extrusion process. The cooling jacket also helps to keep the extrusion temperature constant, which should be approximately 51 ° C (124 ° F). If the mass is too hot (above 165 ° F (74 ° C)), the mass will be damaged.

Uniform flow of dough through the extruder is also important. Variances in the mass flow through the die cause the mass to be extruded at different flows. Non-uniform products should be discarded or re-processed, which increases the unit cost of the product. The surface of the matrix also influences the appearance of the product. Until recently, most matrices were made of bronze, which was relatively soft and required periodic repair or replacement. Recently, dies have been refined by equipping the die extruder surface with Teflon® inserts to extend die life and improve dough quality.

Drying is the most difficult and critical step to control in the mass production process. The purpose of drying is to reduce the moisture content of the dough from approximately 31% to 12 to 13% so that the finished product will be rigid, retain its shape and be stored without spoiling. Most pasta drying operations use a preliminary dryer immediately after extrusion to prevent dough from sticking. Pre-drying hardens the outer surface of the dough while keeping the interior soft and plastic. A final dryer is then used to remove most moisture from the product. Drying temperature and relative humidity increments are important factors in drying. Since the outer surface of the dough dries faster than the inside, moisture gradients develop across the surface into the dough. If dried too quickly, the dough will break, yielding a poor looking product with very low mechanical strength. Disruption may occur during the drying process or as long as several weeks after the product has left the dryer. If the dough is dried too slowly, it tends to spoil or become moldy during the drying process. Therefore, it is essential that the drying cycle is adapted to meet the requirements of each type of product. If the drying cycle has been successful, the dough will be firm but also flexible enough that it can bend to a considerable degree before breaking.

Packaging keeps the product free of contamination, protects the mass from damage during shipping and storage, and displays the product favorably. The main packaging material for noodles is the cellophane bag, which provides moisture proof protection for the product and is easily used on automatic packaging machines, but is difficult to stack on warehouse shelves. Many manufacturers use boxes instead of bags to pack dough because boxes are easy to stack, provide good protection for fragile dough products, and offer the opportunity to print the ad that is easier to read than in bags.

Air emissions can come from a variety of sources in pasta making. Particulate matter (PM) emissions result mainly from solids handling and mixing. For pasta making, PM emissions occur during the wheat milling process, as the raw ingredients are mixed, and possibly during packaging. Emission sources associated with wheat milling include grain receiving, pre-cleaning / handling, house cleaning, milling and bulk loading. Other information is provided by D.E. Walsh and KA Gilles, Pasta Technology, Elements of Food Technology, NW Desrosier, Editor, AVIR Publishing Company, Inc., 1977. The present invention is applicable to both industrial manufacturing and homemade dough preparation, in the latter case favorably. in the preparation of pasta with eggs.

In accordance with the present invention, in the process of preparing pasta, the lactic acid bacterial mixture described herein is used.

In another embodiment of the present invention, a typical Asian cereal-based food is provided. A preferred example is a type of noodle known in Korea as Ramyun, in China as Ramien and in Japan as Ranrten.

As in the general embodiment of the present invention, the pasta is prepared by addition of the lactic acid bacterial mixture and left long enough for pre-fermentation.

The mixtures of lactic acid bacteria and bifidobacteria according to the present invention, optionally added with the above mixtures of microbial proteases, may also be used in the manufacture of a food, in particular gluten free grade, for consumption by an affected patient. for celiac disease. Examples of this type of food are pasta, cereals, tacos, tortillas, popcorn. For reference see Practi-cal Gastroenterology - April 2004, pages 86-104 and the literature cited here.

Another object of the present invention is a method for the manufacture of a baked article comprising the addition of the above sour dough preparation.

In a preferred embodiment of the present invention, the method comprises the following steps: a) liquid pre-fermentation of 20-50% wheat flour by weight (integral mixture of 20% -50% flour and 80% -50% water, mass yield of about 300 with about 109 cells of the mixture of the present invention per gram of mass) at about 37 ° C for at least about 24 hours, preferably between about 24 and about 31 hours; b) after fermentation, mix the dough with one or more tolerated flours, such as millet flour, to have a final dough yield of about 150 (solid dough) and added baker's yeast at a concentration of about 1 wt%; c) incubating the mass at about 37 ° C for about 2 hours until fermentation is complete; d) bake at about 250 ° C for about 20 minutes.

In a second embodiment of the present invention, the method may be modified as follows: a) liquid fermentation of 20% wheat flour with subsequent addition of fungal proteases (200 ppm) at 37 ° C for 24 to 31 hours; (b) after fermentation, dry to remove water to have a gluten-free wheat flour (<200 ppm gluten); (c) use of gluten-free wheat flour as a basic ingredient for the manufacture of cereal-based food, in particular baked goods. The term "fence" in this circumstance means values around those indicated which are understood in the normal execution of the invention and may depend on instrumental errors of the measuring devices or deviations made by persons skilled in the art around the indicated values, but which do not affect the result obtained by the invention.

The above ranges are also intended to be about greater than the lower limit and about smaller than the lower limit. Therefore, the liquid pre-fermentation of step (a) comprises a quantity of wheat flour of not less than 20% and not more than 50% by weight for a time not less than about 24 hours and not more than about 31 hours. .

A list of examples of tolerated flour include bean flour, buckwheat flour, flax flour, corn (maize), leguminous flour (garbanzo / chickpeas, lentil, pea), millet, Indian Grown Rice Grass. , nut flour (almond, hazelnut, pecan), quinoa, potato flour, sweet potato flour, sago, seed (sesame) flour, sorghum, soy, tapioca, teff. Millet is one of the preferred tolerated flours.

A very advantageous embodiment of the present invention is to include a prebiotic in the baked article whether or not it contains a tolerated flour.

A prebiotic is a substance similar to an undigested fiber, examples of which are short and long chain oligosaccharides such as fructooligosaccharides, soybean oligosaccharides, xyloligosaccharides and iso-maltooligosaccharides.

An even more advantageous embodiment of the invention is the incorporation of the baked article described herein into a baked article such as that described in EP 1 010 372. In that embodiment, the baked article comprises an essentially water-free, uncooked fat-based composition. comprising live lyophilized lactic acid bacteria. Such a fat-based composition comprising live lyophilized lactic bacteria can be naturally combined with all baked products of the present invention. The cereal-based foodstuff, in particular cooked articles and the packages for the cereal-based foodstuff being made, in particular cooked articles in accordance with the present invention are suitable for administration to a patient suffering from celiac disease.

As stated above, the present invention also comprises general food known under the generic name starchy food, in particular cereal based food. The lactic acid bacterial mixture, optionally added with the microbial protease as previously described, is used in the manufacture of starchy food, in particular cereal-based food to obtain the same results and advantages of the above described embodiment of baked goods. In other words, the food obtained according to the present invention is suitable for patients suffering from sciatic disease or for general consumers, also in good health, who desire more digestible food. For example, children and older people may want more digestible food.

Therefore, another object of the present invention is a method for treating a patient suffering from sciatic disease comprising integrating said patient's diet into a cooked article and / or a starchy food as described above. As stated above, cooked articles and starchy food according to the present invention will be comprised in the term "cereal based food".

In another embodiment of the present invention, cereal-based food, in particular baked articles, may also be used to maintain gluten tolerance or to induce gluten tolerance or to reduce the risk of allergies due to albumin and globulin from flour. wheat.

In another embodiment of the present invention, cereal based food, in particular cooked foods may be safely used by celiac patients as long as they have low gluten concentration (<200 ppm).

Treatment methods according to the present invention may also be used in combination with other medical treatments for celiac disease.

As reported above, schizophrenic symptoms are observed in celiac patients and schizophrenic patients show gluten-sensitive behavior. The mixtures according to the present invention are useful for preparing gluten free dietary articles.

Therefore, another object of the present invention is the use of the mixture described above in the preparation of a gluten free dietary article useful for the treatment of schizophrenic symptoms. In particular, said symptoms affect a celiac or non-celiac patient.

Another problem in the art is the use of proline in enteral diet preparations. In certain patients, proline is not hydrolyzed and the compounds for preparing the enteral diet solution are not assimilated. Allergic responses to proline may also occur. The bacterial mixtures of lactic acid and bifidobacteria described in the present invention are useful for hydrolyzing proline or proline rich peptides, thereby making effective non-allergic enteric diet preparations.

Thanks to these properties, the mixtures of lactic acid bacteria and bifidobacteria described in the present invention are also useful for preparing hypoallergenic gliadin enriched glutamine solutions.

In another embodiment of the present invention, it has also been found that the mixtures described herein may be used in the manufacture of a product to reduce levels of Platelet Activating Factor (PAF) and other inflammatory cytokines to treat gastrointestinal disease. PAF is involved in a number of gastrointestinal disorders, in particular inflammatory disorders. We may mention ischemic bowel necrosis (Hsue W., Gonzalez-Crussi F.; Methods Achiev, Exp. Pathoir, 1988: 13; 208-39), gastric ulcer (Esplugues JV, Whittle BJ, Methods Find. 1989: Supl. 1, 6106), hemorrhagic retocolitis (Chaussade S., Denizot Y, Ann. Gastroenterol. Hepatol. (Paris); May 1991, 27 (3); 117-21), necrotizing enterocolitis (Ewer AK., Acta Pediatr. Suppl , 2002, 91 (437): 2-5; neonatal: Caplan MS., Et al., Semin Pediatr. Surg .; August 2005, 14 (3): 154-51), inflammatory bowel disease (Nassif A., et al., Dis. Colori Rectum; February 1996 ;; 39 (2): 217-23), bolsite (Rothenberg DA., Et al. Ann. Chir. 1993; 47 (10): 1043- 6).

In view of the above explanation, the product according to the present invention may also be supplemented for patients, particularly the Japanese people, having PAF hydrolase deficiency, which may be affected by a number of inflammatory diseases (Karasawa K., et. al .; Prog. Lipid Res. March 2003, 42 (2): 93-114). The product may take the form of a food as described above, or a nutritional supplement, a nutraceutical, a drug.

Nutritional and nutraceutical supplements are terms well known in the art (Arvanitoyannis IS, et al., Crit. See. Food Sci. Nutr .; 2005,45 (5): 385-404 and Kalra EK, AAPS PharmaScí; 2003, 5 (3 ); E25) and there is no need for further definition. The following example subsequently illustrates the invention.

Example 1 Fermentation of sour dough and electrophoresis analysis The characteristics of the wheat flour used were as follows: moisture, 12.8%; protein (N x 5.70), 10.7% dry matter (DM); fat, 1.8% DM; ashes, 0.6% MS; and total soluble carbohydrates, 1.5% DM. Eighty grams of wheat flour and 190 mL of tap water (containing a cell concentration of cell preparations of about 109 CFU per gram of flour) was used to produce 270 g mass (mass yield, 220). Four masses were made using the following mixtures of lactic acid bacteria and bifidobacteria.

Mixture 1 according to the invention;

Streptococcus thermophilus. Infant Bifidobacterium, Bifidobac-terium longum, Bifidobacterium brevis, Lacotbacillus acidophilus, Lactobacil-lus plantarum, Lacotbacillus casei, Lactobacillus delbrueckki subsp. bulgari-cus;

Mixture 2 according to the invention: Streptococcus thermophilus, Bifidobacterium iactis, Bifidobacterium brevis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus.

Mixture 3: Lactobacillus acidophilus, Lactobacillus brevis, Streptococcus thermophilus, Bifidobacterium infant.

Mixture 4: Lactobacillus brevis, Lactobacillus saiivarius spp. SaUcinius, Lactobacillus plantarum. Fermentation was carried out at 37 ° C for 24 hours. An inoculum-free mass of bacteria was chemically acidified to pH 4.0 with a mixture of lactic acid and acetic acid (4: 1 molar ratio) and used as a control. After incubation, gliadin was extracted from the masses following the method originally described by Osborne (Osborne, TB; 1970, The Proteins of the Wheat Kernel. Carnegie Institute of Washington, publication 84. Judd and Detweiler, Washington, DC) and further modified. by Weiss et al. (Weiss etal.; 1993, Electrophoresis, 14: 805-816).

Aliquots of 10 to 20 µl (about 10 µg gliadin) were diluted 1: 1 with sample buffer, treated at 100 ° C for 5 minutes and analyzed by sodium dodecyl sulfate (SDS-polyacrylamide gel electrophoresis). PAGE) according to the Laemmli procedure (Laemmli; 1970, Nature, 227: 680-685); the gels contained 12% acrylamide and were stained with Coomassie B10 Bio-Safe Blue (Bio-Rad Laboratories, Hercules, CA). Two-dimensional gel electrophoresis (2DE) was performed as described by Di Cagno et al. (Dt Cagno; et al., 2004). Three gels were analyzed, and the spot intensities of chemically modified mass (siCAD) and sour mass (added from MIX 1) (siSD) were normalized as reported by Bini et al. (Bini et al.; 1997, Electrophosis, 18: 2832-2841). The hydrolysis factor for the individual proteins was expressed as [(siCAD - siSD) / siCAD] x 100. All hydrolysis factors were calculated based on the average spot intensities of the three gels and the standard deviation was calculated. Only hydrolysis factors with statistical significance where Pera <0.05 were reported. Hydrolysis of synthetic substrates. Pro-rich Polypeptides and RP-FPLC Analysis

Preliminarily, the protein-specific peptidase activities of Mix 1 were characterized by the use of synthetic substrates such as Pro-p-NA, Leu-p-NA, AIA-p-NA, Leu-Leu, Val-Leu, Pro. -Gly, Gly-Pro-Ala, Leu-Leu-Leu, Z-Giy-Pro-p-NA, and NCBZ-Gly-G! Y-Leu-p-NA (Sigma Chemical Co., St. Louis, Mo ). The assay mixture contained 500 µl 200 mM phosphate buffer, pH 7.5, 150 µl substrate (0.2-3 mM, final concentration), 8 µl NaN3 (0.05% final concentration) and 50 µl. µl of the MIX 1 preparation (5 x 10 9 CFU / mL, final concentration). The A-gliadin fragment 62-75 (PQPQLPYPQPQSFP) (Silano and De Vicenzi; 1999) and the 33-mer epitope (LQLGPFPGPQLPYPQPQLPYP-QPQLP-YP-GPQPF) (Shan et al., 2002) were synthesized by the Neosystem Laborato -rie (Strasbourg, France). The assay mixture for fragment 62-75 contained 320 µl 20 mM phosphate buffer, pH 7.0, 150 µl substrate (450 µM, final concentration), 8 µl NaN3 (0.05% final concentration) and 50 µl of the MIX 1 preparation (5 x 10 9 CFU / mL, final concentration). The 33-mer epitope assay mixture contained 500 µl 200 mM phosphate buffer, pH 7.5, 150 µl substrate (200 µM, final concentration), 8 µl NaN3 (5 x 109 CFU / µl, concentration Final). Both mixtures were incubated at 37 ° C under agitated conditions (150 rpm). Enzyme kinetics for 33-mer hydrolysis were calculated using a Linewea-ver-Burk plot (Lineweaver and Burk; 1934. J. American Chem. Soc., 56: 658-666).

Enzyme reactions were stopped by addition of 0.05% (v / v) (final concentration) trifluoroacetic acid. The peptides were separated from the mixture by RP-FPLC using a 3 mL Resource II RPC column and FPLC equipment with a 214 nm UV detector (Amersham Biosciences, Upssala, Sweden). Elution was at a flow rate of 1 mL / min with a gradient (5 to 100%) of acetonitrile in 0.05% trifluoroacetic acid. CH3CN concentration was linearly increased from 5 to 46% between 16 and 62 minutes and from 46% to 100% between 62 and 72 minutes. The same procedure was used to determine the oligopeptides contained in the 70% ethanol soluble extracts of the fermented masses.

Use of fungal proteases in combination with lactic acid bacteria and bifidobacteria To produce gluten-free sour dough (<200 ppm), MIX 1 was used in combination with 200 ppm of fungal proteases routinely used in bakery products. During fermentation, the complementary activity of the proteolytic activities of bacteria and fungal sources resulted in a marked increase especially of gliadin and glutenin fractions. Ethanol-soluble extracts of the fermented sour dough showed a gluten concentration of less than 200 ppm as determined by the use of the R5 monoclonal antibody.

Western blot analysis with R5 monoclonal antibody and RAPD PCR analysis The fermented mass (37 ° C for 24 hours) of MIX 1 (about 109 CFU per gram of mass) was mixed with non-protein ingredients and toiered flour (for millet) to produce Italian cookies and baked at 250 ° C for 15 minutes. Italian biscuits made without fermentation with MIX 1 were used as controls. The biscuits were analyzed by Western blot with mo-noclonal antibody R5 and by RAPD PCR at the National Biotechnology Center, Gluten Unit (28049, Madrid, Spain). Monoclonal antibody R5 recognizes potential toxic celiac peptides: QQPFP and 33-mer. RAPD PCR was performed based on specific DNA sequences that are related to potential toxic peptides.

Hydrolysis of salt-soluble proteins from wheat flour (albumins and qlobulins). Albumin and globulin were extracted from wheat flour by the method of Weiss (1993). The assay mixture containing 0.8 mL albumin / globulins (about 3 mg / mL) in 50 mM Tris-HCl, pH 7.0, 5 x 10 9 CFU / mL of MIX 1 AND NaN3 at 0 ° C, 05%. Incubation was at 37 ° C for 24 hours under agitated conditions. A control without microbial cells was included in the test. After incubation, the supernatant was recovered by centrifugation and used for electrophoresis. Water / salt soluble fraction proteins (albumin and globulin) were analyzed by immunoblotting (Curioni, A., et al., 1999, Clin. Exp. Allergy, 29: 407-413) to detect IgE binding of pooled sera. of atopic patients, previously characterized as suffering from gastrointestinal symptoms related to wheat ingestion. Using a semi-dry blotting, protein bands, separated by SDS-PAGE, were transferred to nitrocellufose sheets with a Trans-blot Cell (Bio-Rad Laboratories, Milan, Italy) with a transfer buffer containing 48 mM of Tris, pH 9.2, 39 mM glycine, 20% methanol and 0.1% SDS for 5 hours at 50 V voltage. Blotting bands were visualized by soaking the membranes for a few minutes in Ponce-au S (0.1% in 3% trichloroacetic acid) and marked with a pencil before discoloration in water. Membranes were blocked with TBS containing 0.05% Tween 20 (TBS-T) and 5% skimmed milk powder (M-TBS-T) for 2 hours and incubated overnight with pooled patient sera. diluted 1:20 in TBS-T. After five washes with M-TBS-T, the blots were incubated for 1 hour with monoclonal anti-human peroxide conjugated IgE antibody (Sigma Chemical Co), diluted 1: 5,000 in M-TBS-T (Curioni, et al. a!., 1999). After four washes in M-TBS-T and one in TBS, bound IgE was visualized by chemiluminescence using Supersignal Detection kit (Pierce Biotechnology Inc., Rockford, IL) according to the instructions provided by the manufacturer. The procedure was performed at room temperature.

Compared to the control, SDS-PAGE profiles of gliadin fractions extracted from fermented masses with the four cell preparations showed that not all cell preparations had the same ability to degrade gliadin. Hydrolysis was too high for MIX 1 of the invention, only lighter for MIX 2, while the other cell preparations (MIX 3 and 4) did not yield appreciable degradation.

Differences between the four cell preparations were confirmed by RP-FPLC analysis of 70% ethanol-soluble protein fractions which yielded a full view of oligopeptides with apparent molecular masses lower than those detectable by electrophoresis.

The above results gave great evidence of the higher performance of MIX 1 which appeared to have a more specifically gliadin-directed proteolytic activity.

When the bacterial species, which made up MIX 1, were used individually at the same concentration of about 109 cells per gram mass, none of the 8 species yielded marked hydrolysis as shown by the mixture. This was the first evidence of complementary proteolytic activity among species of at least 6 strains that are used in MIX 1 in well-defined proportion.

Gliadin and related oligopeptides are characterized by a large proportion of proline residues within their sequences {Wieser, 1996, Acta Pediatr. Suppl. 412: 3-9). Proline is unique among the 20 amino acids because of its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins, making them extremely resistant to hydrolysis. To properly handle such peptides, a group of specific peptidases is required to hydrolyze all peptide bonds, where proline residue occurs as a potential substrate at different positions (Cunnin-gham and Connor; 1997, Biochim. Biophys. Acta , 1343: 160-186). Preliminarily, MIX 1 proline-specific peptidase activities were characterized by the use of synthetic substrates such as Pro-p-NA, Leu-p-NA, Ala-p-NA, Leu-Leu, Val-Leu, Pro-Gly. , Gly-Pro-Ala, Leu-Leu-Leu, Z-Gly-Pro-p-NA and NCBZ-Glu-Gly-Leu-p-NA which are relatively specific for the proline iminopeptidase, aminopeptidase, dipeptidase, prolinase, prolidase, dipeptidyl peptidase, tripeptidase, prolyl endopeptidase and endopeptidase, respectively (Table 1).

Table 1 Activities of MIXING enzymes 1.

Each value is the average of three enzyme assays, and standard deviations were calculated. One unit of enzyme activity (U) on p-NA substrates was defined as the amount of enzyme that produced an increase in absorbance at 410 of 0.01 / min. One unit in the polypeptides was the amount of enzyme that releases 1 micromol of substrates / minute.

All of the above enzymatic activities have been widely distributed in the preparation of MIX 1. As it is very rare for a single microbial strain to possess all previous enzymatic activities (Cunnmgham and O'Connor; 1997; Kunjii et al .; 1996, Antoine Van Leeuwe -nhoek 70: 187-221; Di Cagno et al.; 2004), only a collection of selected bacteria, such as those contained in MIX 1, can have the complete peptidase pattern required for hydrolysis of Pro-rich oligopeptides. of gliadin oligopeptides by the preparation of MIX 1 during dough fermentation was further characterized by 2DE analysis. Eighty-four protein spots were identified in the chemically acidified mass used as a control (Figure 1A). Seventy-nine of the 84 gliadin oligopeptide patches were markedly degraded after mass fermentation with MIX 1 compared to the control (Figure 1B). Table 2 refers to the spot hydrolysis factors identified by 2DE. Most degraded oligopeptides (65 out of 79) had hydrolysis factors greater than 80% and only 8 showed hydrolysis factors less than 40%.

Table 2 Properties of MIXED 1 hydrolyzed alcohol-soluble polypeptides after incubation of the mass at 37 ° C for 24 hours. Analyzes were performed with the Image Master software (Pharmacia). Four independent replicating gels were analyzed. For blot quantification and hydrolysis factor calculation, see Materials and Methods. All hydrolysis factors were calculated based on the mean spot intensities of each of the four gels, and standard deviations were calculated. bThe blot designation corresponds to those gels in Figures 1A and 1B.

The above results showed that MIX 1 was able to almost completely hydrolyze gliadin oligopeptides. The activity of MIX 1 was further characterized in vitro in relation to some of the oligopeptides reported in the literature as the main responsible for SC; A-gliadin fragment 62-75 (Silano and De Vincenzi; 1999) and the 33-mer epitope (Shan et al.; 2002). As shown by RP-FPLCA analysis, A-gliadin fragment 62-75, at a concentration of 450 μΜ, was completely hydrolyzed after 6 hours incubation with 5 x 10 9 cfu / mL of MIX 1 cells. 33-mer, at a concentration of 200 μΜ, was completely hydrolyzed after 24 h of incubation with the same cell concentration as MIX 1 (Figure 2). The kinetics of 33-mer hydrolysis were determined by Lineweaver-Burk plotting showing a Vmax of 0.26 pmol per milliliter per minute and a Km of 216 μΜ. As previously reported in the literature, it should be noted that the 33-mer epitope has the following properties: (1) it remains intact despite prolonged exposure to gastric and pancreatic proteases; (ii) it shows less than 20% hydrolysis during 20 h incubation with small brush edge membrane enzymes; and (iii) it remains intact for a long time (about 24 h) in the small intestine and even at low concentration acts as a potential antigen for T cell proliferation (Shan, et al., 2002). The above results showed that MIX 1 contained the complex collection of enzymatic activities required to completely hydrolyze 33-mer and that these activities are markedly greater than those located at the gastrointestinal level.

Compared to references to European gliadin, the R5 monoclonal antibody Western blot from Italian biscuits had the typical profile of intact gliadin. A major advantage of the R5 monoclonal antibody is its ability to recognize the consensus amino acid sequence QXPW / FP (Osman, et al.; 2001, Eur. J. Gastroenterol. Hepatol., 13: 1189-1193) corresponding to epitope repeats multiple immunoreactive agents, which occur in α-, γ- and ω-gliadin, as well as in different wheat varieties (Shewry et al.; 1992, Cereal's protein and celiac disease. In: Celiac disease, Marsh M. [ed], Oxford , Blackwell Scientific Publications pp. 305-348). Higher reactivity has been associated with the QQPFP amino acid sequence, but homologous repeats such as LQPFP, QLOPYP, QLPTF, QQSFP, QQTFP, PQPPP, QQPYP, and PQPFP are also recognized with a weaker reactivity to the R5 antibody (Osman, et al. ; 2001). Interestingly, three of these epitopes (LQPFP, QLPYP, and PQPFP) are placed in the potent inducer sequence of human T-cell lines derived from the gut peptide A-gliadin 33-mer (Shan, et al.; 2002 ). The Western blot of Italian cookies fermented with MIX 1 showed a near degradation of the α-, β- and γ-gliadin recognized by the monoclonal antibody.

The same results were confirmed by RAPD PCR analysis.

Preliminary experiments to identify allergenic fractions of wheat albumin and globulin indicated that 100% of the sera tested were positive against albumin and globulin fractions. Responses were verified against protein components with apparent molecular masses of 15 to 70 Kda, with intense staining for some sera around 15 to 45 KDa. As determined by one-dimensional DAS-PAGE, the comparison of untreated albumin and globulin with that hydrolyzed by the MIX 1 preparation enhanced the hydrolysis of several potential allergenic polypeptides.

Example 2 The sour dough prepared according to Example 1 was used in the manufacture of baked goods.

The baked products as described in US Patent Examples 6,884,443 were prepared by using the pasta composition according to the present invention instead of that of the patent. Fermentation was performed at 37 ° C for 24 hours as described in Example 1 above and MIX 1 was used.

The products became more digestible and suitable for patients infected with celiac disease.

Example 3 The sour dough prepared according to Example 1 with MIX 2 was used in the manufacture of the dough. The mass has become more digestible and can be ingested by patients affected by celiac disease.

Example 4 MIX 1 according to Example 1 was used in the manufacture of noodles according to the teaching of US 2002/0160093.

Examples 1-4 of US 2002/01600993 were repeated except that a packet containing MIX 1 according to the present invention was added to the kansui and flour mixture. After kneading, the mixture was allowed to stand at 37 ° C for 24 hours. Then noodles were prepared as described in the reference. The product has become more digestible and suitable for patients affected by celiac disease.

Example 5 MIX 2 according to Example 1 was used in the manufacture of noodles according to the teaching of WO 99/65331.

Examples 1-2 of WO 99/65331 were repeated except for the package containing MIX 2 according to the present invention which was added to the dough ingredient. After mixing, the dough was allowed to stand at 37 ° C for 24 hours. Then noodles were prepared as described in the reference. The product has become more digestible and suitable for patients affected by celiac disease.

Example 6 MIX 1 according to Example 1 was used in the manufacture of bread derivatives according to the teaching of EP 0 614 609.

Examples 1 to 5 of EP 0 614 609 were repeated except that a packet containing MIX 1 according to the present invention was added to the dough preparation. After kneading, the dough was allowed to stand at 37 ° C for 24 hours. The products were then prepared as described in the reference. The product has become more digestible and suitable for patients affected by celiac disease.

Example 7 MIX 2 according to Example 1 was used in the manufacture of spaghetti according to the teaching of EP 1 338 209. Example 1 of EP 1 338 209 was repeated except that a packet containing a MIX 2 of according to the present invention has been added to the dough ingredient. After mixing, the dough was allowed to stand at 37 ° C for 24 hours. Then noodles were prepared as described in the reference. The product has become more digestible and suitable for patients affected by celiac disease.

Example 8 Ramyun Composition: 1. Noodles Flour: 83-85% Refined Oil: 15-18% Salt: 1% Others: 0.6-1% 2. Dried Soup Base Dried Steak Flake, Soy Sauce, Monosodium Glutamate , Disodium Glutamate, Flavoring Additive, Glucose, Garlic, Onion, Green Onion, Red Pepper Powder, Other Flavoring Ingredient.

Ramvun Manufacturing Process Flour (sometimes starch, rice flour, barley flour may be used for a different reason) and water are mixed according to the manufacturer's recommendation. MIX 1 was added to the batter and allowed to stand at 37 ° C for 24 hours.

The roll was passed into the dough mixed with pressure roller and then the dough was put through the machine to make the noodle strips. The shape and thickness of the noodle may be modified to the desired thickness and shape by adjusting the size of the stripper slot and also the speed of the noodle conveyor belt. The noodles passed through the steam box whose temperature is higher than 100 ° C to induce pregelatinized starch (-starch) to make digestion easier.

After steaming, the noodles are formed in the shape determined by the molding box.

Deep Frying Process: Depending on the type of Ramyun, dehydration occurs through the deep frying process. The noodles pass through the deep frying process at 150 ° C. Some Ramyun does not go through this deep frying process.

After the deep frying process, Ramyun stops through the cooling process.

In accordance with the present invention, the specific mixture of lactic acid bacteria and bifidobacteria is suitable for the manufacture of cereal based food, in particular cooked articles, which may be better tolerated by SC patients. In particular, the following advantages are provided: (i) enhanced ability to degrade gliadin oligopeptides during fermentation; (ii) hydrolysis of 79 of the 84 gliadin oligopeptides identified by 2DE analysis; (iii) complementary and large enzyme activities with respect to synthetic peptides that included proline in different positions; (iv) ability to completely hydrolyze oligopeptides (A-gliadin fragment 62-75 and epitope 33-mer), which are responsible for CS; (v) ability to significantly reduce α ·, β- and γ-gliadin that reacted with monoclonal antibody R5; (vi) ability to hydrolyze various allergenic polypeptides; (vii) when the activity of the above bacteria is supplemented with fungal proteins and used with respect to 20% wheat flour under liquid fermentation, it greatly increases the production of gluten free wheat flour.

Claims (35)

Sour pasta, characterized in that it comprises a mixture of at least 6 species of lactic acid bacteria and / or bifidobacteria selected from the group consisting of: Lactobacillus aci-dophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus plantarum, Bifidobacterium brevis, Bifidobacterium infantil, Bifidobacterium longum, Streptococcus thermophi-lus, Bifidobacterium lactis, in which at least one species of lactic acid bacterium and at least one bifidobacterium and enzymatic protein is present. which has a gluten concentration of less than 200 ppm. Sour dough according to Claim 1, characterized in that said mixture of lactic acid bacteria and bifidobacteria comprises Streptococcus thermophillus, Infant Bifidobacterium, Bifidobacterium longum, Bifobacterium brief, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus casei subsp. bulgaricus, or said mixture comprises Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium brevis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus. Sour dough according to claim 1, characterized in that said proteolytic enzyme is of fungal or bacterial source. Sour dough according to claim 3, characterized in that said proteolytic enzyme is obtained from Aspergillus sp. Fermentation composition, characterized in that it comprises a mixture of at least 6 species of lactic acid bacteria and / or bifidobacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus plantarum, Bifidobacterium brevis, Bifidobacterium infantil, Bifidobacterium longum, Streptococcus thermophilus, Bifidobacterium lactis, in which at least one species of lactic acid bacterium and at least one bifidobacterial enzyme and protein is present, a gluten concentration less than 200 ppm. Fermentation composition according to claim 5, characterized in that said mixture of lactic acid bacteria and bifidobacteria comprises Streptococcus thermophillus, Infant Bifidobacterium, Bifidobacterium longum, Bifobacterium brevis, Lactobacilius aci-dophilus, Lactobacilius casearilius, , Lactobacilius delbru-eckii subsp. bulgaricus, or said mixture comprises Streptococcus ther-mophilus, Bifidobacterium lactis, Bifidobacterium brevis, Lactobacilius aci-dophilus, Lactobacilius casearum, Lactobacilius casei, Lactobacilius helveti-cus. Fermentation composition according to claim 5, characterized in that said proteolytic enzyme is of fungal or bacterial source. Fermentation composition according to claim 7, characterized in that said proteolytic enzyme is obtained from Asper-gillus sp. Use of the sour dough as defined in any one of claims 1 to 4 or the fermentation composition as defined in any one of claims 5 to 8, characterized in that it is in the manufacture of a baked article. 10. Starch food, in particular a cereal-based food, comprising a mixture of at least 6 species of lactic acid bacteria and / or bifidobacteria selected from the group consisting of Lactobacilius acidophilus, Lactobacilius casei, Lactobacilius delbrueckii subsp. bulgaricus, Lactobacilius helveticus, Lactobacilius plantarum, Bifidobacterium brevis, Bifidobacterium infantil, Bifidobacterium longum, Streptococcus thermophiius, Bifidobacterium lactis, in which at least one species of lactic acid bacterium and at least one bifidobacterial enzyme and protein is present, a gluten concentration less than 200 ppm. Food according to Claim 10, characterized in that the mixture of lactic acid bacteria and bifidobacteria comprises Streptococcus thermophillus, Infant Bifidobacterium, Bifido-bacterium longum, Bifobacterium brief, Lactobacillus acidophiius, Lactobaci-llus piantarum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgari-cus, or said mixture comprises Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium brevis, Lactobacillus acidophiius, Lactobacillus piantarum, Lactobacillus casei, Lactobacillus helveticus. Food according to Claim 10 or 11, characterized in that it is a cooked food. Food according to claim 12, characterized in that it is selected from the group consisting of cookies, pastries, cakes, pies, pizza, crackers, breadsticks, snacks, pasta, noodles, tortillas, corn slices, extruded cereals and strip cereals, ramyun. Cooked article according to claim 10, characterized in that said proteolytic enzyme is of fungal or bacterial origin. Cooked article according to claim 14, characterized in that said proteolytic enzyme is obtained from Aspergillus sp. Packaging for a baked article, characterized in that it comprises the usual ingredients and a fermentation composition as defined in any one of claims 5 to 8. Process for the preparation of a baked article, characterized in that it comprises adding the sour dough preparation as defined in any one of claims 1 to 4 to the usual ingredients. Process according to claim 17, characterized in that it comprises the following steps: a) liquid pre-fermentation of about 20 to 50% by weight wheat flour (whole mixture of 20% to 50% of flour) and 80% to 50% water, mass yield of about 300) with about 109 cells of the mixture as described in any one of claims 1 to 5 per gram of mass at 37 ° C for at least about 24 hours, preferably from about 24 to about 31 hours; (b) after fermentation, mix the dough with one or more tolerated flours, such as millet flour, to have a final dough yield of 150 (solid dough), and add commercial baker's yeast to a concentration of about 1 wt%; c) incubating the mass at about 37 ° C for about 2 hours until fermentation is complete; d) bake at 250 ° C for about 20 minutes. Process according to Claim 18, characterized in that in step b) the dough is mixed with the tolerated flour. Process according to Claim 19, characterized in that the tolerated flour is selected from the group consisting of bean meal, buckwheat, flaxseed, maize flour, millet meal, Rice Grass Grown in India, nut flour, quinoa, potato meal, sweet potato meal, sago, seed meal, sorghum, soybean, tapioca, teff. Process according to any one of claims 17 to 20, characterized in that it further comprises the addition of fungal proteases. Process according to claim 21, when dependent on any one of claims 19 to 21, characterized in that, in step a), said addition is made at 37 ° C and fermented for 24 to 31 hours and Wheat flour is 20% by weight. Baked article, characterized in that it is obtainable by the process as defined in any one of claims 17 to 22. Baked article, in particular according to claim 23, characterized in that it contains one or more tolerated flours. Cooked article according to claim 24, characterized in that said tolerated flour is selected from the group consisting of bean flour, buckwheat, flaxseed, maize, leguminous flour, millet, Rice Grass Grown in India, nut flour, quinoa, potato meal, sweet potato meal, sago, seed meal, sorghum, soybean, tapioca, teff. Food according to any one of claims 10 to 15 or 23 to 25, characterized in that it further comprises a prebiotic. Food as defined in any one of claims 10 to 15 or 23 to 25, characterized in that it is combined with an essentially water-free fat-based composition comprising lyophilized lactic acid bacteria. Gluten-free product, or a low gluten product, characterized in that it is as defined in any one of claims 10 to 15 or any one of claims 23 to 25. Enteric diet product, characterized in that it comprises a mixture as described in any one of claims 1 to 4. Use of sour dough as defined in any one of claims 1 to 4 and / or the fermentation composition as defined in any one of claims 5 to 8, characterized in that it is in the manufacture of a foodstuff, in particular an article. cooked for the integration of the diet of a patient suffering from celiac disease. Use of sour dough as defined in any one of claims 1 to 4 and / or the fermentation composition as defined in any one of claims 5 to 8, characterized in that it is in the manufacture of a food, in particular a food. cooked to maintain gluten tolerance in a patient suffering from celiac disease. Use of the sour dough as defined in any one of claims 1 to 4 and / or the fermentation composition as defined in any one of claims 5 to 8 in the manufacture of a food, in particular a food cooked to induce gluten tolerance in a patient suffering from celiac disease. Use of the sour dough as defined in any one of claims 1 to 4 and / or the fermentation composition as defined in any one of claims 5 to 8, characterized in that it is in the manufacture of a food, in particular an article. cooked to reduce the risk of allergies due to wheat flour albumins and globulins. Use of the mixture as defined in any one of claims 1 to 4, characterized in that it is in the preparation of a dietary article useful for the treatment of schizophrenic symptoms in a celiac patient. Use of the mixture as defined in any one of claims 1 to 4, characterized in that it is in the preparation of a product for oral administration to improve digestion of gluten and gluten related substances.